Integrated metabolomics and mass spectrometry imaging analysis reveal the efficacy and mechanism of Huangkui capsule on type 2 diabetic nephropathy

Background: Huangkui capsule (HKC), a Chinese patent medicine, is clinically used for treating diabetic nephropathy. However, the core disease-specific biomarkers and targets of type 2 diabetic nephropathy (T2DN) and the therapeutic mechanism of HKC are not fully elucidated.

Purpose: This study aimed to investigate the therapeutic effects and underlying molecular mechanisms of HKC for T2DN.

Study design: The db/db mouse model was used to evaluate the efficacy of HKC for T2DN, and the core pathways regulated by HKC were studied to determine its kidney protective mechanism.

Methods: High-throughput UPLC-MS/MS and multivariate analysis were employed to analyze the serum and kidney metabolic profiles of db/db mice, identifying potential core biomarkers of T2DN. Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry imaging was used to locate in situ spatial distribution of core biomarkers and drug active ingredients in kidney tissues. Biochemical indicators, histopathology, immunohistochemistry, immunofluorescence, molecular docking, and western blotting were combined to reveal therapeutic effects, pathways, and targets of HKC.

Results: HKC substantially improved pathological characteristics, kidney function, oxidative stress, inflammation, and lipid metabolism indicators of T2DN. Twelve core disease-specific biomarker that significantly influenced clustering were identified and its unique spatial distribution information in the kidneys was revealed. 3-dehydrosphinganine, retinyl ester, and 9-cis-retinoic acid (9cRA) could serve as novel disease-specific biomarkers for T2DN. Based on newly discovered biomarkers, quercetin, myricetin, and isorhamnetin were found to act on key enzymes SPT, ALDH1A1, AOX, LRAT, and DGAT1 in retinol and sphingolipid metabolism pathways. Western blotting showed that HKC ameliorated T2DN by targeting these enzymes, upregulating 9cRA and retinyl ester, downregulating 3-dehydrosphinganine, increasing TGF-β signal transduction, inhibiting the expression of the immune fibrosis proteins OX-8, Col-I and α-SMA, inhibiting Th17 cell development and ceramide synthesis, reducing IL-1β, TNF-α, MDA, TC, LDL-C, and TG levels, and increaseing SOD activity.

Conclusions: HKC exerts significant therapeutic effects on T2DN. HKC corrects the metabolic disorder of sphingolipids and retinol, and improves T2DN by regulating the activities of SPT, ALDH1A1, AOX, LRAT, and DGAT1. This study provides valuable ideas and new mechanistic insights for the treatment of T2DN with HKC.